Respiratory Distress Syndrome (RDS) is a progressive respiratory failure in newborns caused by inadequate surfactant function. Approximately 26,000 infants died of RDS during the years 1975 to 1978 and many others suffered significant short- and long-term morbidity as a result of the disease and its therapy annually. The average hospital costs are about 2.5 times higher for premature infants with RDS than without. Thus, there are both medical and economic reasons for exploring new possibilities for simple and reliable prediction of RDS, as well as effective and safe therapies for the disease.
The need to assess in particular the status of the surfactant, a complex mixture predominantly of phospholipids with smaller amounts of protein, in pregnancies in which fetal well-being is jeopardized by maternal diabetes mellitus, toxemia, induced labor or delivery by Caesarian section is commonly recognized. There are no known clinically applicable methods of directly quantitating surfactant in the alveoli and airways, but fortunately substitute methods are available. Fluid continuously moves out of the lungs before birth and carries surfactant into the amniotic fluid. Surfactant components can be measured in the fluid, and have a good correlation with pulmonary surfactant.
Currently, the most accepted clinical test has been the amniotic fluid lecithin/sphingomyelin (L/S) ratio. The L/S assay uses centrifugation of the fluid to remove cell debris, lipid extraction with chloroform-methanol mixture, removal of "non-surfactant" lecithin by cold acetone precipitation and separation of phospholipids by one or two dimensional thin layer chromatographic procedures. The ratio of L/S is determined by either densiometry of the chromatogram to measure the intensity of each spot; by planimetry to estimate their area; or by phosphorus analysis, to determine the amount of each lipid.
Helena L/S ratio kit (Helena Fetal-Tek 200) marketed by Helena Laboratories, 1530 Lindbergh Dr. P.O. Box 752, Beaumont, Tex. 77704, is based on this technique. A L/S ratio of 2.0 which represents a mature value is basically reliable when performed according to a strictly prescribed procedure. However, technical expertise and the strictest quality control measures are necessary to successfully perform the time consuming procedure and correctly interpret the data. In addition, a number of abnormal conditions encountered in pregnancy such as diabetes mellitus, Rh isoimmunization, maternal hypertension, premature deliveries or membrane rupture, as well as contamination of the amniotic fluid with blood or meconium, are known to cause altered lecithin values which may give a false indication of lung maturity in the fetus, thus resulting in large number of false predictions. There are several modifications of this technique which use some form of lipid extraction followed by separation on thin layer chromatography and then lipid determination. All of them have limitations similar to those attributed to the L/S method.
Fluorescence polarization measurement of amniotic fluid is also used for determination of fetal lung maturity. Fluorescence polarization is a measure of the microviscosity of the whole lipid assembly of amniotic fluid and claimed to reflect an overall property, not yet fully understood, of the total lipid mixture present in the fluid. Fluorescence polarization values correlate with changes in L/S ratio but also suffer from blood contamination in addition to intrinsic problems associated with fluorescence measurements. Finally, the fluorescence polarization method requires very expensive instrumentation.
There are some rapid assays such as the shake test and the absorbance measurement at 650 nm which demonstrate some correlation with L/S ratio. The shake test or the foam stability index is based on the presence of surfactant in amniotic fluid to form a stable foam in the presence of ethanol. These assays are straightforward and inexpensive techniques. However, their predictive values are relatively low and are affected by any contamination in the fluid. In addition, they do not measure specific elements of surfactant in the fluid. Therefore, they are mostly used as screening assays and in combination with other assays.
Recently, it has been recognized that two specific phospholipids, phosphatylglycerol (PG) and phosphatidylinositol (PI), are very important to the biological process of lung maturation and that their determination in amniotic fluid aids in prediction of RDS. It is believed that PG analysis predicts the level of maturity more accurately than the L/S test. The presence of PG, even with an L/S ratio indicating lung immaturity predicts that the fetus will be free of RDS. PG and PI are present in blood in very small amounts. Therefore, assays detecting their presence in amniotic fluid will not suffer from contamination with blood.
In most normal pregnancies, PI increases after 30 weeks of gestation and peaks at 36-37 weeks. PG increases after 35 weeks and continues to increase beyond term. Initially, only Thin Layer Chromatography (TLC) methods were available for PG analysis. As in the case regarding TLC analysis of L/S ratios, the technique is subject to many variables, is difficult to perform and is very time consuming. In addition, PG determination requires additional steps beyond those needed for L/S ratios.
Because of these problems, several other assays for PG were proposed. Hana Biologics, Inc., 626 Bancroft Way, Berkley, Calif. 94710, has commercialized a biochemical immunologic agglutination test for determining the presence of PG (Amniostat-FLM). The assay is said to be specific, sensitive and fast. The results of the tests are not affected by blood or meconium contamination of fluid samples. However, since the assay is expressed as a negative, positive or strong positive for the presence of PG, it can be used only as a screening test, or as one in a series of tests performed to obtain a more thorough assessment of fetal lung maturity. A major limitation of this procedure is that it relies on biological reagents which are labile and require careful storage conditions. In addition, the agglutination reaction is temperature dependent and not all specimens will produce agglutination patterns identical to the equivalent control reaction. Special care has to be taken when looking for "clearing in the background" of the agglutination reaction, otherwise a false positive interpretation may be obtained. Recently, a new enzymatic method for quantitating PG in amniotic fluid has been offered by ISOLAB Inc., Akron, Ohio.
There is no commercial procedure, however, which incorporates the determination of PI for evaluating lung maturation. There is also a growing number of investigators and hospitals who assay for more than one phospholipid when assessing fetal pulmonary maturity. Thus the sum of all surface active phospholipids, a combination of the L/S ratio and PG determination and the total phospholipid concentration in the lamellar body fraction of amniotic fluid are being used.
In summary, the current methods used in prediction of lung maturity suffer from one or more of the following disadvantages: they require technical expertise and expensive instrumentation; they are time-consuming; they lack specificity and sensitivity and use unstable biological reagents, and/or they are at best only semiquantitative. All this is reflected in inconsistent data and large expenses per test.
Therefore, a great need exists for a rapid, simple, inexpensive, and accurate test for the evaluation of the lipid content of biological fluids, for example, the evaluation of amniotic fluid, which information is used in the prediction of fetal pulmonary maturity.